During clinical anesthesia, despite dramatic losses of neural function many parts of the nervous system remain active. The relatively specific loss of consciousness and memory make volatile general anesthetics (VGAs) inviting tools for studying these higher functions. And, anesthesiologists would like to use rational drug design to limit the dangerous side effects of these drugs while retaining their efficacy. However, identifying the cellular and molecular basis for anesthesia has been frustrating. This is largely because communication within the nervous system depends on the proper functioning of a large number of components and, even at clinically relevant concentrations, VGAs interfere with a surprisingly large number of these entities. As a means to identify anesthetic targets, we have undertaken the isolation and characterization of randomly generated mutations that perturb the response to VGAs of the fruit fly, Drosophila melanogaster. Our past experience has established three important points. First, flies resemble people in their overall sensitivity to VGAs. Second, at doses of VGAs that cause gross behavioral effects, some parts of the flys nervous system remain functional. Third, mutations that alter the flys responses to VGAs can be readily isolated. This experience suggests that the anesthetic targets are conserved, specific and non-essential. However, our collection of mutants is small and, because they were obtained by chemical mutagenesis, they have been hard to clone. Accordingly, we have undertaken a transposon mutagenesis for anesthesia mutants. To date, we have isolated more than 500 sex-linked random insertions of a marked mobile element, Pz. Each of these has been screened with halothane in the distribution test, developed in this lab as a rapid and reliable way to measure concentration dependence of anesthetic effects on a simple behavior. This identified more than twenty independent lines with significant shifts in the dose-response curve. Ongoing work is establishing both the linkage of the anesthetic effect to the Pz element and the DNA sequence that flanks the insertion site. The new collection will be a rich source of readily manipulable genetic variation in loci that influence the potency of VGAs. A major problem for our research is that, in addition to altering a target molecule, mutations can alter anesthetic sensitivity indirectly. For example, genetic variation could influence the activity of an anesthetic-insensitive pathway that parallels an anesthetic-sensitive pathway, both of which can execute a particular behavior. The problem is compounded because assays of complex behavior typically involve elements of the nervous system that are manifold and largely unknown, making it hard to pinpoint mutational effects. How then can one use genetics to identify the cellular and molecular targets of general anesthetics? We think that the best strategy is to use behavioral assays like the distribution test to identify mutants with altered sensitivity to anesthetics and then to screen among these mutants with physiological and/or biochemical assays on identified cells and/or components that respond directly to VGAs. Toward this end, we have undertaken the determination of anesthetic effects on the electroretinogram (ERG) of Drosophila. The ERG is a mass potential recorded from the surface of the eye that reports electrical activity in the photoreceptors and in the large monoplar cells (LMCs) they innervate. Our results show that neither the photoreceptor response to light nor the initial transmission to the postsynaptic cell in response to a light-on signal is perturbed by clinical doses of VGAs. But, the light-off response of LMCs is quite sensitive to VGAs. This suggests that local feedback from cells innervated by LMCs is sensitive to VGA action. Such cells have been identified anatomically and, at least in larger flies, physiologically. We therefore believe that ERG effects offer our best tool to date to screen amongst anesthesia mutants. - Drosophila melanogaster, general anesthetics, visual system, P element, halothane